JP3017887B2 - Methods for inactivating prionene, viruses and other infectious agents - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the inactivation of prionene, viruses and other infectious agents, such as bacteria, present in plasma or serum which are suitable as starting materials for obtaining biological or biogenic substances, in particular albumin. How to do it.

[0002]

BACKGROUND OF THE INVENTION In preparing proteins from animal (or human) material, there is always the risk of contamination by infectious particles that were present in the starting material. A recent common example is the transmission of many humans with the HIV-virus (the pathogen of AIDS), for example, by administration of blood products to hemophiliac patients. These infections were before the discovery of the AIDS factor, in particular, and donors have been examined or screened since then. In addition, manufacturers are exploring ways to remove HIV-viruses, which may be present in the donor's blood, during serum protein purification and mass production of therapeutics.

[0003] As the nature of this virulence factor is elucidated,
It is thought that it would be possible to develop a method for disintegrating this more easily and quickly. For bacteria, the first infectious particles found in history, there are several well-known methods for decades (steam sterilization, dry heat sterilization, low temperature sterilization, filtration sterilization, ethylene oxide sterilization, radiation sterilization, etc.). All have reached a certain technical level.

[0004] Although there is a method for removing a virus (various group of pathogenic factors) first discovered in the twentieth century, a specific method for all pathogenic factors has not been found yet. Although many viruses cause complete disintegration when the pH is lowered to 4, some viruses are inactivated by high concentrations of organic solvents.

[0005] A few years ago, a new group of virulence factors emerged that cast doubt on molecular biology theories and began to appear in academic journals. This factor is called prionene. Although the physical properties of this prionene are not yet clear, some scientists consider prionene to be a very small virus,
Some consider small rod-shaped nucleic acids with several coat proteins. On the other hand, most scientists have come to the groundbreaking conclusion that prionene is related to infectious proteins without DNA or RNA, since prionene cannot prove the presence of the nucleic acid. This is nucleic acid (genetic material)
Is the first objection to the belief that is essential for the replication of infectious particles.

[0006] The nature of prionene remains largely unknown, and only a few such diseases are known to humans or animals, but since there is no cure for it, this disease is a fatal disease. is there. Research and creation of therapeutics is extremely difficult because the incubation period is related to infectious diseases spanning 30 years, but consumption is expected to increase. One of these diseases is BSE (bovine spongiform encephalopathy)-an unexplained bovine epidemic that occurs in the UK.
Common and very similar diseases include scrapie in sheep and goats (probably the current source of BSE disease).

[0007] In the case of humans, there are kuru (disease seen at festivals in Papua New Guinea), Jacob-Creuzfeld syndrome and Gerstadtmann-Straussler syndrome. Fortunately, these rare diseases have a low incidence, on the order of one millionth, but are very similar to Alzheimer's disease.

[0008] There is evidence that BSE was widespread in the United Kingdom through the administration of animal feed from animal processing facilities that disposed of scraped sheep. Unfortunately, there is a corresponding disease in humans, due to the incorporation of Creutzfeld-Jakob syndrome pathogens at the manufacturer's responsibility in producing human growth hormone (secretory substance) from the human pituitary gland. It is. Many of these diseases have already been expressed in patients receiving this hormone, and have been observed in young people up to 50 years of age.

[0009] Potential dangers are pointed out from this new type of disease. Poor knowledge of this new virulence factor, prionene, makes it just as difficult to assess the risk for treatment. It is also possible that prionene has almost no nucleic acid, and that the prionene protein is transformed into a pathogen after being encoded by a host. In addition, prionene does not generate an immune response such as antibody formation, and is very difficult to diagnose.

Attempts have been made to destroy prionene, but there is one major problem. That is, prionene exhibits extremely strong resistance to all physical and chemical methods, but is preferably treated with a high concentration of a mineral acid or alkali solution at a high temperature, or with a hypochlorite-alkali solution. , Or it is known to be removed by temperatures above 150 ° C. However, it is clear that such a powerful treatment will immediately destroy, or at least lose effect, any biological therapeutics.

[0011] Until now, in order to prepare prionene-free biological materials, it has been attempted to obtain starting materials from an area where the above-mentioned infectious diseases have not occurred, but the incubation period is extremely long, 30 years. Due to the length, this method is considered to still carry significant risks. Since no immune response is generated as described above, screening for starting materials cannot confirm the presence of prionene.
Biological tests (injection of target substance into mouse brain, evaluation for pathological response, mouse dissection, molecular biological and histological findings, and confirmation by re-injection into mouse brain) It is time consuming and heavily used, so it is suitable not only for starting material control but also for the overloading test of the finished drug, which is a new production method (after adding prionene). It is also useful for checking effectiveness. Experimental animals are bred and tested (P3
Zone), must be performed under secure equipment in special laboratories.

[0012]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to destroy all known virus groups without decomposing biological substances or biogen substances, and to exist in these substances. It is to provide a method for reliably removing prionene.

[0013]

DISCLOSURE OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and has a constitution comprising a plasma suitable as a starting material for obtaining any biological substance or biogen substance, particularly albumin. Alternatively, a method of inactivating prionene, virus and other infectious agents present in serum with a chaotropic reagent, wherein the substance is treated with a chaotropic reagent or diluted with sterile pyrogen-free water at 1 g / l. Add surfactant and add 7
After stirring at a temperature of 0 ° C., the resulting solution is treated with the chaotropic reagent at room temperature for 12 to 18 hours.

That is, it can be said that treating prionene with a chaotropic reagent at room temperature (20 to 25 ° C.) for a long time is epoch-making. This type of treatment does not degrade, for example, biological materials such as albumin. Also,
The treatment ensures that viruses and bacteria are removed.

As the chaotropic reagent, it is preferable to add 6 to 8 mol of urea based on the whole amount, or 1 to 2 mol of sodium thiocyanate based on the whole amount. After treatment, these reagents can be removed by dialysis, membrane filtration or gel filtration chromatography.

To increase the certainty of removing the pathogenic agent, the biological or biogen material is adjusted to a pH of about 6.5 before treatment with the chaotropic reagent or after dilution with sterile pyrogen-free water, to a pH of about 1 g / g. It is preferred to add 1 surfactant, especially an anionic surfactant. As the surfactant, alkyl sulfate or a derivative thereof,
Preferably, sodium alkyl sulfate and / or sarcosinate, preferably sodium (N-lauryl sarcosinate), and / or any of alkyl sulfonates or derivatives thereof or a mixture thereof can be added. If necessary, the pH value is adjusted by the administration of dilute hydrochloric acid.

Further, it is preferable to add 8 to 10% by volume of methyl alcohol or ethyl alcohol.

Finally, after the addition of the surfactant, optionally methyl alcohol or ethyl alcohol, the biological or biogenic substance is heated at about 70 ° C. with slow stirring, at this temperature for at least 15 minutes, preferably about
The mixture is further stirred for 30 minutes, after which it is cooled rapidly and the pH is lowered to 4-4.2 to separate out the precipitated globulin.

In the method of the present invention, for example, bovine serum albumin (BSA) is selected as the albumin. Because it is used in large quantities and the source of material "bovine" is endangered by BSE, and because its human analogue, human serum albumin (HSA), is a very important therapeutic agent It is. The method can be diverted directly to the production of HSA without modification, and the method can modify other proteins of animal or human origin. For example, serum and IgG can be mentioned.

An example in which the method of the present invention is applied to BSA is as follows. After diluting the plasma or serum from which BSA or HSA is obtained with water, the pH value is adjusted to 6.5 and a strong surfactant, preferably sodium lauryl sulfate (sodium dodecyl sulfate) or an analogue, sodium- In the presence of (N-lauryl sarcosinate) or other analogues (1 g / l), add ethyl alcohol or methyl alicol and stir at a temperature of 70 ° C. for 15-30 minutes. Thereafter, the pH is immediately adjusted to 4.0-4.2 to remove the precipitated globulin and neutralize the clear solution.

High concentrations of strong chaotropic reagents (preferably 6-8 moles of urea or sodium thiocyanate 1
２2 mol), the solution is left at room temperature for 16 hours, after which the reagent can be removed, preferably by chromatography, dialysis or membrane filtration.
Then, the salt-free solution is concentrated and then added with an additive, sterilized by filtration, and transferred to another container in a sterilized state, or
In particular, it is made to dry by freeze-drying. The thermal post-treatment of the solution ("pasteur sterilization" in the presence of stabilizers) or of the powder (steam injection) can be combined.

In addition, instead of the above-mentioned serum or plasma,
Any other starting material for which albumin is present, for example,
Placental blood and a fraction obtained by plasma fractionation by the cone method are also used.

Thus, the efficacy was determined by adding scrapie / prionene to the source plasma in a biological experimental model,
And by injection of the albumin solution into the mouse brain. Similarly, conventional viruses such as bovine viral diarrhea (BVD) virus, bovine infectious rhinotracheitis virus, type 3 pararifleenza virus, foot and mouth disease virus, retro-visna virus, and sheep parapoxvirus are It has also been demonstrated by administration at high concentrations. The albumin solution thus treated was self-sterilizing, and the bacteria were destroyed.

[0024]

Next, an embodiment of the present invention will be described. 3g
Of bovine lyophilized plasma (equivalent to 2 g of protein) was dissolved in 35 ml of distilled sterilized water, and 4 ml of ethanol immiscible with water was added thereto, and the pH was adjusted to 6.5 with dilute hydrochloric acid.
(= 0.1%) sodium laurate (at least 9% pure)
5%) was dissolved. The total volume was 44 ml. to this,
Sufficient safety facilities (P3 level laboratory, laminar flow hood, etc.)
225 μl of 20% scrapie / brain / homogenate (titer: 2.10 LD / g) or a highly concentrated virus solution was added thereto to homogenize.

This solution was placed in a 70 ° C. water bath. After 10 minutes, the temperature of the contents was likewise 70 ° C., and the above treatment was continued for another 30 minutes. The mixture was stirred with a magnetic stirrer for 1 minute every 10 minutes. At the end of the incubation, the sample is transferred to ice and cooled, and the cooled solution is
The pH was adjusted to 4.2 with 0 μl of hydrochloric acid and homogenized.

This is 6000 rpm (4000 g) at 4 ° C. for 10 minutes.
Globulin) was removed by centrifugal force. The clear supernatant was obtained by decanting and was a pure bovine albumin solution (volume: 20.5 ml).

The solution was neutralized (pH 7.0) by adding 250 μl of a sodium hydroxide solution. Thereafter, 15 g of urea was added to increase the volume to 30 ml, resulting in a concentration of 8 molar. These solutions were left at room temperature (21 ° C.) for 16 hours.

For the removal of urea, gel filtration chromatography was used as follows. 15 g of Sephadex G50 (Pharmacia, Uppsala) dissolved in 500 ml of sterile distilled water was heated and left overnight. The swollen gel was packed in an acrylic column having a diameter of 5 cm and a height of 30 cm, and washed with sterilized distilled water (200 ml). The flow rate was 7.5 ml / min.
30 ml of this solution was applied to a column and washed with 100 ml of water. The obtained fraction was divided into 50 ml and 40 ml. After that,
No protein was observed in the eluate. This 90 ml was freeze-dried to obtain 0.7 g of bovine serum albumin.

In some experiments, the Amicon S-1 model (cutoff value: 10,
The urea was removed by membrane filtration at 000d). At that time, about
The membrane was filtered with 1000 ml of distilled water, concentrated to about 50 ml, and then lyophilized.

Next, various biological tests will be described. The results of these tests were as shown in Table 1. Biological test for scrapie / BSE: The above albumin was dissolved in 3 ml of saline and 20 μl of the reagent was injected into each half of the mouse brain. The total number of mice was 136, and the strain was C57 / B16. This scrapie / inoculum (titer: 2.10 LD / g), ie, an 8-fold dilution was used as a positive control, and bovine serum albumin as above prepared without scrapie / inoculum was used as a negative control. did. All inoculations were performed one by one with 12 animals. That is, it has been repeated 12 times. The results of this test showed that the positive control was ill, whereas the negative control did not show any illness or death.

Biological test using conventional virus: The above BSA was dissolved in a cell culture solution. This medium is used as a nutrient medium for mammalian cells, which can be host cells for specific viruses. The test was performed by adding positive (virus strain solution) and negative (medium only) controls, and a non-specific cytotoxicity test for albumin. The results show that no infectious virions were able to survive in any of the test virus strains, and with this objective method the mortality exceeded 1/10 ⁶ (parts per million).

In a similar test, N-lauryl sarcosinate and lauryl sulphate were shown to be equally effective as surfactants in the preparation of albumin from plasma, but the administration of infectious substances Did not do. The parameters measured were the yield and purity of BSA with both surfactants above 98%.

In treating influenza virus ribonucleoprotein-particles (experimental model for examining nucleic acid-protein interactions) and dissociating affinity bonds such as gelatin-fipronectin, an equivalent amount of chaotropic reagent is used. Urea and sodium thiocyanate have been tried to prove their effectiveness.

[0034]

[Table 1]

[0035]

The present invention has been described above, and it is intended to remove prionene, virus and other infectious agents present in plasma or serum, which are suitable as starting materials for obtaining any biological substance or biogen substance, especially albumin. A method of inactivating with a chaotropic reagent, wherein before the substance is treated with the chaotropic reagent or diluted with sterile pyrogen-free water, 1 g / l of a surfactant is added, and 70 ° C.
After stirring at a temperature of 12 ° C., the resulting solution is treated with a chaotropic reagent at room temperature for 12 to 18 hours to inactivate the biological solution. The group can be disintegrated, and the prionene present in these substances can be easily and reliably removed.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification code FI A61P 31/12 A61P 31/12 (56) Reference Proc. Natl. Acad. Sc i. USA Vol. 79, no. 17 (1982) p5220-5224 (58) Fields investigated (Int. Cl. ⁷ , DB name) A01N 61/00 WPI (DIALOG)

Claims (9)

(57) [Claims] 1. A method for inactivating with a chaotropic reagent any prionene, virus and other infectious agents present in plasma or serum which are suitable as starting materials for obtaining any biological or biogenic substance, in particular albumin. Before treating the substance with a chaotropic reagent or diluting with sterile pyrogen-free water, adding 1 g / l of a surfactant and stirring at a temperature of 70 ° C., the obtained solution was treated with the chaotropic reagent. A method for inactivating prionene, virus and other infectious agents, which is carried out at room temperature for 12 to 18 hours. 2. The method according to claim 1, wherein urea is used as a chaotropic reagent in a concentration of 6 to 8 mol based on the total amount. 3. The method according to claim 1, wherein sodium thiocyanate is used as a chaotropic reagent in a concentration of 1 to 2 mol based on the total amount. 4. The method according to claim 1, wherein the chaotropic reagent is removed after treatment by dialysis, membrane filtration or gel filtration chromatography. 5. Before the treatment with a chaotropic reagent or after dilution with sterile pyrogen-free water, the pH of the biological substance or biogen substance is adjusted to a weak acidity of 6.5, and 1 g / l of an anionic surfactant is added. 2. The method according to claim 1, wherein said additive is added.
5. The method according to any one of items 1 to 4. 6. As a surfactant, alkyl sulfate or a derivative thereof, or sodium alkyl sulfate and / or sarcosinate, or sodium (N-lauryl sarcosinate) and / or alkyl sulfonate or a derivative thereof is used. 6. The method according to claim 5, wherein the addition is performed. 7. The method according to claim 5, wherein the pH value is adjusted by administering dilute hydrochloric acid. 8. The method according to claim 5, wherein 8 to 10% by volume of methyl alcohol or ethyl alcohol is added. 9. After addition of the surfactant, optionally methyl alcohol or ethyl alcohol, the biological or biogenic substance is heated with gentle stirring at 70 ° C. and further stirred at this temperature for 15 to 30 minutes, 9. The process according to claim 1, wherein the globulins are rapidly cooled and the pH is lowered to a strongly acidic pH of 4 to 4.2 to separate the precipitated globulin.